Project Summary Most opioid analgesics used clinically, including morphine and fentanyl, act primarily through the mu opioid receptors. However, opioid analgesia and side-effects, such as constipation, itch, sedation and addiction, vary immensely among patients and animal models. These observations suggest the existence of multiple mu opioid receptors, a concept that was initially proposed based on pharmacological studies and has been reinforced by molecular cloning of a vast array of mu opioid receptor variants, generated through extensive alternative pre-mRNA splicing from the single-copy mu opioid receptor (OPRM1) gene. Increasing evidence indicates that the OPRM1 splice variants are important in mediating the complex and variable actions of mu opioids. Two distinct promoters, associated with either exon 1 (E1) or exon 11 (E11), control the expression of E1-associated or E11-associated splice variants. E1-associated variants mainly encode full-length carboxyl (C- ) terminal, 7-transmembrane (7-TM) domain receptors, whereas the majority of E11-associated variants encode truncated, 6-TM domain receptors. Gene targeted mouse models have revealed that E1-associated full-length 7-TM C-terminal variants mediate the actions of morphine and methadone, whereas E11-associated truncated 6-TM variants mediate a subset of mu opioids, including fentanyl, M6G and heroin, as well as IBNtxA (3-iodobenzoyl-6?-naltrexamide), a novel analgesic that lacks many traditional opioid side-effects. While the mouse models are valuable, rats have many advantages both in behavioral modeling and in vivo manipulation. Rats have been used extensively to study opioid actions for many decades, many of which have not been conducted in mice. Rats are considered as better models than mice in a number of human conditions. Having rat models would provide significant advantages and resources for the research community, and would expand our ability explore mu opioid pharmacology with approaches and techniques not feasible in mice. Splicing in the rat OPRM1 gene is similar to that in the mouse and human OPRM1 genes, with an extensive array of both E1-associated and E11-associated variants. Yet, the lack of appropriate rat gene targeting models limits further exploration of their functions. We propose generating three rat OPRM1 knockout/knockin (KO/KI) models, E1- KO/KI, E11-KO/KI and E1/E11-KO/KI, using recently developed CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 nuclease) technology. We will characterize E1-KO/KI and E11- KO/KI models by examining the expression of OPRM1 splice variants, the analgesic actions of selected opioids, as well as the endogenous E1 and E11 promoter activity. These mutant rat models will provide unique and valuable tools for the research community to further explore the pharmacological functions and transcriptional regulation of the rat OPRM1 gene. These studies will allow us to obtain a better understanding of the complex actions of various opioids, and to design novel, potent opioid analgesics that lack traditional opioid side-effects and abuse potential.